Difference between revisions of "Part:BBa K2856002"
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===Usage and Biology=== | ===Usage and Biology=== | ||
| − | S-adenosyl-methionine synthetase encoded by gene metK is an enzyme involved and responded to the synthetic reaction of S-adenosyl-methionine (SAM). In this reaction, one molecule methionine and one molecule ATP are converted to one molecule SAM (Figure 1). The L. lactis NZ9000 has the ability to synthesize SAM, but just enough for itself. In our project, we constructed a plasmid harboring metK in order to produce more SAM in L. lactis. | + | <br> |
| + | S-adenosyl-methionine synthetase encoded by gene metK is an enzyme involved and responded to the synthetic reaction of S-adenosyl-methionine (SAM). In this reaction, one molecule methionine and one molecule ATP are converted to one molecule SAM (Figure 1). The L. lactis NZ9000 has the ability to synthesize SAM, but just enough for itself. In our project, we constructed a plasmid harboring metK in order to produce more SAM in L. lactis.<br><br> | ||
| − | [[File:T--H14Z1 Hangzhou--Reaction_metK.jpeg|800px|thumb|centre| <p>'''Figure. 1 Enzymatic reaction catalyzed by metK'''</p>]] | + | [[File:T--H14Z1 Hangzhou--Reaction_metK.jpeg|800px|thumb|centre| <p>'''Figure. 1 Enzymatic reaction catalyzed by metK'''</p>]]<br><br> |
=== Construction and validation of plasmid pNZ-metK === | === Construction and validation of plasmid pNZ-metK === | ||
| + | <br> | ||
Gene metK was amplified from genomic DNA of Lactococcus lactis NZ9000 and cut with restriction enzyme Hind III and KpnI, and ligased with plasmid pNZ8148 cut with the same enzyme. Then the ligation product was transferred to E.coli and spread on plates containing 10 mg/L chloramphenicol. | Gene metK was amplified from genomic DNA of Lactococcus lactis NZ9000 and cut with restriction enzyme Hind III and KpnI, and ligased with plasmid pNZ8148 cut with the same enzyme. Then the ligation product was transferred to E.coli and spread on plates containing 10 mg/L chloramphenicol. | ||
| − | Colonies on the plates were randomly picked and inoculated in 1ml LB medium for 3 hours at 37℃, 200 rpm. 1 μl culture were added to the PCR system as template. As shown in Figure. 2, all the picked colonies had gene metK, illustrating that the plasmid pNZ-metK was successfully constructed. | + | Colonies on the plates were randomly picked and inoculated in 1ml LB medium for 3 hours at 37℃, 200 rpm. 1 μl culture were added to the PCR system as template. As shown in Figure. 2, all the picked colonies had gene metK, illustrating that the plasmid pNZ-metK was successfully constructed. <br><br> |
| − | [[File:T--H14Z1 Hangzhou--Plasmid_metK.jpeg|300px|thumb|centre| <p>'''Figure. 2 Validation of plasmid pNZ-metK. M represented marker. 1-5 represented three randomly picked colonies.'''</p>]] | + | [[File:T--H14Z1 Hangzhou--Plasmid_metK.jpeg|300px|thumb|centre| <p>'''Figure. 2 Validation of plasmid pNZ-metK. M represented marker. 1-5 represented three randomly picked colonies.'''</p>]]<br><br> |
=== Protein Analysis === | === Protein Analysis === | ||
| − | After transferring the plasmid pNZ-metK to L. lactis NZ9000, SDS-PAGE was performed to detect the protein expression level of metK gene. The cells were washed twice with 0.1 M PBS after centrifugation. Crude protein was extracted through cell breaking using ultrasonication and centrifugation. Then the supernatant of the samples were used to analysis the protein expression. As shown in Figure. 3, expected bands of the MetK protein were observed on the gel. Recombinant L. lactis containing pNZ-metK induced with different nisin concentration showed higher expression of MetK protein. | + | <br> |
| − | [[File:T--H14Z1 Hangzhou--SDS-PAGE_metK.jpeg|400px|thumb|centre| <p>'''Figure. 3 SDS PAGE validation of gene metK expression in L. lactis. M represented marker. WT represented L. lactis NZ9000. 1-3 represented L. lactis/pNZ-metK induced with 100 50 and 20 ng/ml nisin.'''</p>]] | + | After transferring the plasmid pNZ-metK to L. lactis NZ9000, SDS-PAGE was performed to detect the protein expression level of metK gene. The cells were washed twice with 0.1 M PBS after centrifugation. Crude protein was extracted through cell breaking using ultrasonication and centrifugation. Then the supernatant of the samples were used to analysis the protein expression. As shown in Figure. 3, expected bands of the MetK protein were observed on the gel. Recombinant L. lactis containing pNZ-metK induced with different nisin concentration showed higher expression of MetK protein.<br><br> |
| + | [[File:T--H14Z1 Hangzhou--SDS-PAGE_metK.jpeg|400px|thumb|centre| <p>'''Figure. 3 SDS PAGE validation of gene metK expression in L. lactis. M represented marker. WT represented L. lactis NZ9000. 1-3 represented L. lactis/pNZ-metK induced with 100 50 and 20 ng/ml nisin.'''</p>]]<br><br> | ||
===Validation of S-adenosyl-methionine (SAM) by HPLC analysis === | ===Validation of S-adenosyl-methionine (SAM) by HPLC analysis === | ||
| − | To confirm the synthetic S-adenosyl-methionine in L. lactis/pNZ-metK, HPLC was performed to analyze the extracts from the strain. S-adenosyl-methionine was identified on the basis of retention times related to standard sample. According to the retention time of standard S-adenosyl-methionine sample, it can be confirmed that S-adenosyl-methionine was synthesized more in L. lactis/pNZ-metK than original L. lactis NZ9000. | + | <br> |
| − | [[File:T--H14Z1 Hangzhou--HPLC_SAM.jpeg|700px|thumb|centre| <p>'''Figure. 4 Validation of S-adenosyl-methionine (SAM) by HPLC'''</p>]] | + | To confirm the synthetic S-adenosyl-methionine in L. lactis/pNZ-metK, HPLC was performed to analyze the extracts from the strain. S-adenosyl-methionine was identified on the basis of retention times related to standard sample. According to the retention time of standard S-adenosyl-methionine sample, it can be confirmed that S-adenosyl-methionine was synthesized more in L. lactis/pNZ-metK than original L. lactis NZ9000.<br><br> |
| + | [[File:T--H14Z1 Hangzhou--HPLC_SAM.jpeg|700px|thumb|centre| <p>'''Figure. 4 Validation of S-adenosyl-methionine (SAM) by HPLC'''</p>]]<br><br> | ||
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
Revision as of 12:04, 17 October 2018
Modification of S-adenosylmethionine synthetase (MetK)
The BBa_K2856001 harbors a coding sequence of S-adenosyl-methionine synthetase (MetK) derived from Lactococcus lactis NZ9000 genome. The MetK protein catalyzes methionine and ATP to form S-adenosyl-methionine.
Usage and Biology
S-adenosyl-methionine synthetase encoded by gene metK is an enzyme involved and responded to the synthetic reaction of S-adenosyl-methionine (SAM). In this reaction, one molecule methionine and one molecule ATP are converted to one molecule SAM (Figure 1). The L. lactis NZ9000 has the ability to synthesize SAM, but just enough for itself. In our project, we constructed a plasmid harboring metK in order to produce more SAM in L. lactis.
Construction and validation of plasmid pNZ-metK
Gene metK was amplified from genomic DNA of Lactococcus lactis NZ9000 and cut with restriction enzyme Hind III and KpnI, and ligased with plasmid pNZ8148 cut with the same enzyme. Then the ligation product was transferred to E.coli and spread on plates containing 10 mg/L chloramphenicol.
Colonies on the plates were randomly picked and inoculated in 1ml LB medium for 3 hours at 37℃, 200 rpm. 1 μl culture were added to the PCR system as template. As shown in Figure. 2, all the picked colonies had gene metK, illustrating that the plasmid pNZ-metK was successfully constructed.
Protein Analysis
After transferring the plasmid pNZ-metK to L. lactis NZ9000, SDS-PAGE was performed to detect the protein expression level of metK gene. The cells were washed twice with 0.1 M PBS after centrifugation. Crude protein was extracted through cell breaking using ultrasonication and centrifugation. Then the supernatant of the samples were used to analysis the protein expression. As shown in Figure. 3, expected bands of the MetK protein were observed on the gel. Recombinant L. lactis containing pNZ-metK induced with different nisin concentration showed higher expression of MetK protein.
Validation of S-adenosyl-methionine (SAM) by HPLC analysis
To confirm the synthetic S-adenosyl-methionine in L. lactis/pNZ-metK, HPLC was performed to analyze the extracts from the strain. S-adenosyl-methionine was identified on the basis of retention times related to standard sample. According to the retention time of standard S-adenosyl-methionine sample, it can be confirmed that S-adenosyl-methionine was synthesized more in L. lactis/pNZ-metK than original L. lactis NZ9000.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]